RESUMO
Thymidine phosphorylase (TP; EC 2.4.2.4) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KThymidine = 284 ± 55, KPi = 5.8 ± 1.9, KThymine = 244 ± 69, and KdRib-1-P = 90 ± 33 µM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its Km. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the EnzymeâPi complex than the free enzyme, but had better affinity to the free enzyme than the EnzymeâThymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.
Assuntos
Fígado/enzimologia , Timidina Fosforilase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fosfatos/metabolismo , Ribosemonofosfatos/metabolismo , Ribosemonofosfatos/farmacologia , Especificidade por Substrato , Timidina/metabolismo , Timidina Fosforilase/antagonistas & inibidores , Timina/metabolismo , Timina/farmacologiaRESUMO
Malaria remains a significant public health problem worldwide with an estimated annual global incidence of 200 million and an estimated 450,000 annual deaths. Among the five known human malarial species, Plasmodium falciparum is the deadliest and most resistant to antimalarials. Hence, there is a need for new antimalarial targets. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and their hosts. In view of their high rate of replication, parasites require very active nucleic acid synthesis which necessitates large supplies of the indispensable pyrimidine nucleotides. Consequently, delineation of P. falciparum pyrimidine metabolic pathways may reveal potential targets for the chemotherapy of malaria. Previous studies reported the existence of pyrimidine de novo pathways in this organism. The present results demonstrate the presence of enzymes of the pyrimidine salvage pathways in P. falciparum and indicate that this parasite is capable of pyrimidine salvage. Furthermore, some of the pyrimidine salvage enzymes, e.g., dTMP kinase, phosphoribosyltransferase, and uridine phosphorylase could be excellent targets for chemotherapeutic intervention against this parasite.
Assuntos
Plasmodium falciparum/metabolismo , Pirimidinas/metabolismo , Animais , Antimaláricos/farmacologia , Citidina Desaminase/metabolismo , Citosina Desaminase/metabolismo , DCMP Desaminase/metabolismo , Desenho de Fármacos , Eritrócitos/parasitologia , Humanos , Malária Falciparum/sangue , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Redes e Vias Metabólicas , Núcleosídeo-Fosfato Quinase/metabolismo , Pentosiltransferases/metabolismo , Fosfotransferases/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Pirimidina Fosforilases/metabolismoRESUMO
Thymidine phosphorylase (TP; EC 2.4.2.4) is involved regulation of intra- or extracellular thymidine concentration, angiogenesis, cancer chemotherapy, radiotherapy, as well as tumor imaging. Although the liver is main site of pyrimidine metabolism and contains high levels of TP, nonetheless, purification and characterization of human hepatic TP has not been accomplished. We here report the purification and characterization of native human hepatic TP. The enzyme was purified to apparent homogeneity by a procedure shorter and more efficient than previously reported methods. Human hepatic TP has an apparent Kthymidine of 285 ± 55 µM. Like the enzyme from other tissues, it is highly specific to 2'-deoxyribosides. However, in contrast to TP from other normal tissues, the hepatic enzyme is active in the phosphorolysis of 5'-deoxy-5-fluorouridine, and the riboside 5-fluorouridine. Furthermore, native hepatic TP exists in different aggregates of 50 kDa subunits, with unknown aggregation factor(s) while TP from extra tissues exists as a homodimer. Isoelectric point was determined as 4.3. A total of 65 residues in the N-terminal were sequenced. The sequence of these 65 amino acids in hepatic TP has 100% sequence and location homology to the deduced amino acid sequence of the platelet derived-endothelial cell growth factor (PD-ECGF) cDNA. However, and contrary to PD-ECGF, the N-terminal of hepatic TP is blocked. The block was neither N-formyl nor pyrrolidone carboxylic acid moieties. The differences in substrate specificities, existence in multimers, and weak interaction with hydroxyapatite resin strongly suggest that hepatic TP is distinct from the enzyme in normal extrahepatic tissues. These results may have important clinical implications when TP is involved in activation or deactivation of chemotherapeutic agents in different tissues.
Assuntos
Fígado/enzimologia , Timidina Fosforilase/química , Plaquetas/enzimologia , Humanos , Ponto Isoelétrico , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Timidina Fosforilase/isolamento & purificação , Timidina Fosforilase/fisiologiaRESUMO
Schistosomes are responsible for the parasitic disease schistosomiasis, an acute and chronic parasitic ailment that affects >240 million people in 70 countries worldwide. It is the second most devastating parasitic disease after malaria. At least 200,000 deaths per year are associated with the disease. In the absence of the availability of vaccines, chemotherapy is the main stay for combating schistosomiasis. The antischistosomal arsenal is currently limited to a single drug, Praziquantel, which is quite effective with a single-day treatment and virtually no host-toxicity. Recently, however, the question of reduced activity of Praziquantel has been raised. Therefore, the search for alternative antischistosomal drugs merits the study of new approaches of chemotherapy. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and host. Pyrimidine metabolism is an excellent target for such studies. Schistosomes, unlike most of the host tissues, require a very active pyrimidine metabolism for the synthesis of DNA and RNA. This is essential for the production of the enormous numbers of eggs deposited daily by the parasite to which the granulomas response precipitates the pathogenesis of schistosomiasis. Furthermore, there are sufficient differences between corresponding enzymes of pyrimidine metabolism from the host and the parasite that can be exploited to design specific inhibitors or "subversive substrates" for the parasitic enzymes. Specificities of pyrimidine transport also diverge significantly between parasites and their mammalian host. This review deals with studies on pyrimidine metabolism in schistosomes and highlights the unique characteristic of this metabolism that could constitute excellent potential targets for the design of safe and effective antischistosomal drugs. In addition, pyrimidine metabolism in schistosomes is compared with that in other parasites where studies on pyrimidine metabolism have been more elaborate, in the hope of providing leads on how to identify likely chemotherapeutic targets which have not been looked at in schistosomes.
Assuntos
Pirimidinas/biossíntese , Schistosoma/metabolismo , Esquistossomose/metabolismo , Animais , Praziquantel/uso terapêutico , Esquistossomose/tratamento farmacológico , Esquistossomicidas/uso terapêuticoRESUMO
Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo=0.002±0.0002 mM, KATP=0.05±0.008 mM, and Vmax=920±35 µmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.
Assuntos
Adenosina Quinase/metabolismo , Adenosina/metabolismo , Cinética , Toxoplasma/metabolismo , Adenosina/química , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Fosforilação , Especificidade por Substrato , Toxoplasma/enzimologiaRESUMO
Competition studies and column chromatography demonstrated that adults Schistosoma mansoni contains three nucleoside kinases that can phosphorylate pyrimidine nucleosides; a non-specific deoxyriboside kinase (EC 2.7.1.145), a specific uridine kinase and a specific cytidine kinase. The non-specific deoxyriboside kinase can phosphorylate all naturally occurring pyrimidine and purine 2'-deoxyribosides. Uridine and cytidine kinases are specific for uridine and cytidine, respectively. Various nucleoside 5'-triphosphate act as phosphate donors for the three kinases albeit to different degrees. Nucleoside kinases are critical in the activation of potential anti-parasitic drugs which may be identified among the numerous available pyrimidine nucleoside analogues. The finding that S. mansoni have nucleoside kinases that differ from their host enzymes raises the possibilities that certain pyrimidine nucleoside analogues could be selectively toxic to schistosomes.
Assuntos
Fosfotransferases/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Schistosoma mansoni/enzimologia , Animais , Fosforilação , Especificidade por SubstratoRESUMO
An adenine nucleoside phosphorylase (ANP, EC none) activity was identified and partially purified from extracts of Schistosoma mansoni by chromatofocussing column chromatography and molecular sieving. The enzyme is distinct from purine nucleoside phosphorylase (PNP, EC 2.4.2.1). ANP is specific for adenine nucleosides which includes adenosine analogs modified in the aglycone, pentose or both moieties. (e.g. 2'-deoxyadenosine, 5'-deoxy-5'-methylthioadenosine, 5'-deoxy-5'-iodo-2-fluoroadenosine, etc.) The enzyme is also distinct from the mammalian 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP, EC 2.4.2.28) in that it is able of the phosphorolysis of 2'-deoxyadenosine while mammalian MTAP cannot. Because of ANP unique substrate specificity, the enzyme could play a role as a target for chemotherapy of these parasites. Cytotoxic analogs may be designed as subversive substrates that are selectively activated only by the schistosomal ANP.
Assuntos
Adenina/metabolismo , Pentosiltransferases/isolamento & purificação , Pentosiltransferases/metabolismo , Schistosoma mansoni/enzimologia , Animais , Especificidade por SubstratoRESUMO
Potent anti-retroviral therapy has transformed HIV-1 infection into a chronic manageable disease; however, drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study, we utilized a combination of structure-based analysis of cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket that showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell-based biological assays. Using these methods, we obtained the first-generation mimetic drugs and tested these compounds on HIV-1 long terminal repeat-activated transcription. Using biological assays followed by similar in silico analysis to find second-generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the second-generation mimetic against various viral isolates and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2(-/-)γc(-/-) with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Células Cultivadas , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Feminino , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host toxicity and enhancing its chemotherapeutic efficacy against human breast tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UP, EC 2.4.2.3), the enzyme responsible for uridine catabolism. METHODS: SCID mice bearing MDA-MB-468 and MCF-7 human breast tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24, and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg), or their combination was administered orally two or 4 h after FUra injection. Another four administrations of PTAU plus uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. RESULTS: Administration of the maximum tolerated dose (50 mg/kg) of 5-fluorouracil (FUra) to SCID mice bearing human breast MDA-MB-468 and MCF-7 adenocarcinoma tumor xenografts reduced tumor weight by 59 and 61%, respectively. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not rescue from FUra host toxicity as all the mice died. Administration of 120 mg/kg PTAU resulted in partial rescue from this lethal dose of FUra as 38% of inoculated mice survived and the tumor weights were reduced by approximately 67%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra. All of the mice survived, and MDA-MB-468 and MCF-7 tumor weights were reduced by 97% and total remission, respectively. Doubling the FUra treatment dose to 400 mg/kg in the MDA-MB-468 inoculated mice, with the administration of the adjuvant combination treatment of PTAU plus uridine, was unsuccessful in rescuing from FUra toxicity as all the mice died. Lowering the dose of FUra to 300 mg/kg, under the same conditions, resulted in 67% mice survival, and the MCF-7 tumor weights were reduced by 100%. Treatment with uridine alone did not protect from FUra toxicity at 200, 300, and 400 mg/kg as all of the mice died. At the higher dose of 300 and 400 mg/kg FUra, PTAU alone had no rescuing effect. There was no significant difference between MDA-MB-468 and MCF-7 in their response to the different regimens employed in this study in spite of the fact that MDA-MB-468 is estrogen receptor negative while MCF-7 is estrogen receptor positive. CONCLUSIONS: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combination treatment in raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al. in Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the massive doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side effects associated with such doses of uridine. The combination may also allow the escalation of FUra doses for better chemotherapeutic efficacy against human breast carcinoma, with the possibility of avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Animais , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Camundongos , Camundongos SCID , Tiouracila/administração & dosagem , Tiouracila/análogos & derivados , Uridina/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.
Assuntos
Plaquetas/metabolismo , Movimento Celular , Membrana Corioalantoide/irrigação sanguínea , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Ribosemonofosfatos/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Inativação Gênica , Humanos , Integrina alfaV/metabolismo , Integrina alfaVbeta3/metabolismo , Integrina beta3/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Trombina/metabolismo , Timidina Fosforilase/antagonistas & inibidores , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Fatores de Tempo , Uridina Fosforilase/antagonistas & inibidores , Uridina Fosforilase/genética , Uridina Fosforilase/metabolismoRESUMO
Toxoplasma gondii adenosine kinase (EC 2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Several 6-benzylthioinosines have already been identified as subversive substrates of the T. gondii but not human adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not its host. In the present study, we report the testing of the metabolism of several carbocyclic 6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the carbocyclic 6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the carbocyclic 6-benzylthioinosine analogues showed a selective anti-toxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. These results indicate that the oxygen atom of the sugar is not critical for substrate binding. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were carbocyclic 6-(p-methylbenzylthio)inosine (IC(50)=11.9 microM), carbocyclic 6-(p-methoxybenzylthio)inosine (IC(50)=12.1 microM), and carbocyclic 6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=12.8 microM). These compounds have about a 1.5-fold better efficacy relative to their corresponding 6-benzylthioinosine analogues (Rais et al., Biochem Pharmacol 2005;69:1409-19 [29]). The results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that carbocyclic 6-benzylthioinosines are potential anti-toxoplasmic agents.
Assuntos
Adenosina Quinase/metabolismo , Toxoplasma/enzimologia , Toxoplasma/metabolismo , Animais , Feminino , Compostos Inorgânicos/metabolismo , Compostos Inorgânicos/uso terapêutico , Compostos Inorgânicos/toxicidade , Inosina/metabolismo , Inosina/uso terapêutico , Inosina/toxicidade , Ligantes , Camundongos , Nucleotídeos/metabolismo , Nucleotídeos/uso terapêutico , Nucleotídeos/toxicidade , Compostos Orgânicos/metabolismo , Compostos Orgânicos/uso terapêutico , Compostos Orgânicos/toxicidade , Tioinosina/análogos & derivados , Toxoplasmose/tratamento farmacológicoRESUMO
Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.
Assuntos
Adenosina Quinase/antagonistas & inibidores , Tioinosina/análogos & derivados , Toxoplasma/enzimologia , Animais , Desenho de Fármacos , Inosina/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Tioinosina/síntese química , Tioinosina/química , Tioinosina/farmacologiaRESUMO
Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-benzylthioinosines act as subversive substrates of T. gondii, but not human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Moreover, 7-deazaadenosine (tubercidin) was shown to be an excellent ligand of T. gondii adenosine kinase. Therefore, we synthesized 7-deaza-6-benzylthioinosine, and analogues with various substitutions at their phenyl ring, to increase the binding affinity of the 6-benzylthioinosines to T. gondii adenosine kinase. Indeed, the 7-deaza-6-benzylthioinosine analogues were better ligands of T. gondii adenosine kinase than the parent compounds, 6-benzylthioinosine and 7-deazainosine. Herein, we report the testing of the metabolism of these newly synthesized 7-deaza-6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the 7-deaza-6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the 7-deaza-6-benzylthioinosine analogues showed a selective antitoxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were 7-deaza-6-(p-methoxybenzylthio)inosine (IC(50)=4.6 microM), 7-deaza-6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=5.0 microM), and 7-deaza-6-(p-cyanobenzylthio)inosine (IC(50)=5.3 microM). These results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that 7-deaza-6-benzylthioinosines are potential antitoxoplasmic agents.
Assuntos
Adenosina Quinase/metabolismo , Antiprotozoários/uso terapêutico , Tioinosina/metabolismo , Tioinosina/toxicidade , Toxoplasma/enzimologia , Toxoplasmose/tratamento farmacológico , Animais , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Prepúcio do Pênis/patologia , Humanos , Recém-Nascido , Masculino , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Tioinosina/análogos & derivados , Tioinosina/uso terapêutico , Toxoplasma/efeitos dos fármacosRESUMO
Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza- p-cyano-6-benzylthioinosine (IC 50 = 5.3 microM) and 7-deaza- p-methoxy-6-benzylthioinosine (IC 50 = 4.6 microM), were evaluated in cell culture to delineate their selective toxicity.
Assuntos
Adenosina Quinase/antagonistas & inibidores , Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Tioinosina/análogos & derivados , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Adenosina Quinase/metabolismo , Animais , Antiprotozoários/química , Células Cultivadas , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/análogos & derivados , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Tioinosina/síntese química , Tioinosina/química , Tioinosina/farmacologiaRESUMO
We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.
Assuntos
Hipóxia-Isquemia Encefálica/enzimologia , Degeneração Neural/enzimologia , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Uridina Fosforilase/metabolismo , Uridina/farmacologia , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Infarto Encefálico/enzimologia , Infarto Encefálico/fisiopatologia , Infarto Encefálico/prevenção & controle , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiopatologia , Técnicas de Cocultura , Citoproteção/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glucose/deficiência , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Degeneração Neural/fisiopatologia , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Uridina Fosforilase/efeitos dos fármacos , Uridina Fosforilase/genéticaRESUMO
PURPOSE: Thymidine phosphorylase (TP, EC 2.4.2.4) activity varies in different human cancer cell lines. Nevertheless, little is known about the regulatory mechanisms of TP expression in such cancers. Promoter methylation of dinucleotide cytosine-guanine (CpG) sites is a known mechanism of reversible gene expression silencing. METHODS: TP promoter methylation was investigated in five cancer cell lines (SKBR-3, 786-O, HT-29, MDA-231, DLD-1). TP mRNA levels were determined by real-time quantitative PCR. The degree of methylation was identified by bisulfite sequencing. Minimal TP promoter activity was determined by Luciferase reporter assays. DNA-protein interactions were evaluated by electrophoretic mobility shift assays. RESULTS: SKBR-3 cells exhibited the highest TP expression, 786-O, HT-29, and MDA-231 cells exhibited intermediate TP expression, while DLD-1 cells did not express TP as demonstrated by TP mRNA, protein, and enzyme activity levels. SKBR-3 lacked methylation in the TP promoter, intron 1 and exon 1 regions, while DLD-1 showed extensive methylation. Treatment of DLD-1 and SKBR-3 with the methylation-inhibitor, 5-aza-2'-deoxycytidine (5-aza-2dC), resulted in a concentration-dependent increase in TP mRNA and protein levels in DLD-1 but not SKBR-3 cells. Trichostatin-A treatment, a histone deacetylase inhibitor, improved the 5-aza-2dC-induced TP re-activation. Electrophoretic mobility shift assays demonstrated that methylation significantly inhibits transcription factor binding. Supershift analyses suggest that the Sp1 and Sp3 (to a lesser degree) transcription factors have a role in the regulation of TP expression. CONCLUSIONS: These findings suggest that TP promoter methylation is a mechanism for down-regulation of TP expression in cancer cells and may have implications in modulating prognosis of cancer patients.
Assuntos
Metilação de DNA/efeitos dos fármacos , Neoplasias/enzimologia , Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Timidina Fosforilase/antagonistas & inibidores , Timidina Fosforilase/genética , Western Blotting , Linhagem Celular Tumoral , Primers do DNA , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfitos/metabolismo , Timidina Fosforilase/biossínteseRESUMO
Toxoplasma gondii is an intracellular parasitic protozoan that infects approximately a billion people worldwide. Infection with T. gondii represents a major health problem for immunocompromised individuals, such as AIDS patients, organ transplant recipients, and the unborn children of infected mothers. Currently available drugs usually do not eradicate infection and as many as 50% of the patients do not respond to this therapy. Furthermore, they are ineffective against T. gondii tissue cysts. In addition, prolonged exposure to these drugs induces serious host toxicity forcing the discontinuation of the therapy. Finally, there is no effective vaccine currently available for the treatment of toxoplasmosis. Therefore, it is necessary to develop new and effective drugs for the treatment and management of toxoplasmosis. The rational design of a drug depends on the exploitation of fundamental biochemical or physiological differences between pathogens and their host. Some of the most striking differences between T. gondii and their mammalian host are found in purine metabolism. T. gondii, like most parasites studied, lack the ability to synthesize purines do novo and depend on the salvage of purines from their host to satisfy their requirements of purines. In this respect, the salvage of adenosine is the major source of purines in T. gondii. Therefore, interference with adenosine uptake and metabolism in T. gondii can be selectively detrimental to the parasite. The host cells, on the other hand, can still obtain their purine requirements by their de novo pathways. This review will focus on the broad aspects of the adenosine transport and the enzyme adenosine kinase (EC 2.7.1.20) which are the two primary routes for adenosine utilization in T. gondii, in an attempt to illustrate their potentials as targets for chemotherapy against this parasite.
Assuntos
Adenosina/metabolismo , Antiprotozoários/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose/tratamento farmacológico , Toxoplasmose/parasitologia , Sequência de Aminoácidos , Animais , Antiprotozoários/química , Humanos , Dados de Sequência Molecular , Toxoplasma/enzimologia , Toxoplasma/genética , Toxoplasmose/genéticaRESUMO
Toxoplasma gondii is an opportunistic pathogen responsible for toxoplasmosis. T. gondii is a purine auxotroph incapable of de novo purine biosynthesis and depends on salvage pathways for its purine requirements. Adenosine kinase (EC.2.7.1.20) is the major enzyme in the salvage of purines in these parasites. 6-Benzylthioinosine and analogues were established as "subversive substrates" for the T. gondii, but not for the human adenosine kinase. Therefore, these compounds act as selective anti-toxoplasma agents. In the present study, a series of N(6)-benzyladenosine analogues were synthesized from 6-chloropurine riboside with substituted benzylamines via solution phase parallel synthesis. These N(6)-benzyladenosine analogues were evaluated for their binding affinity to purified T. gondii adenosine kinase. Furthermore, the anti-toxoplasma efficacy and host toxicity of these compounds were tested in cell culture. Certain substituents on the aromatic ring improved binding affinity to T. gondii adenosine kinase when compared to the unsubstituted N(6)-benzyladenosine. Similarly, varying the type and position of the substituents on the aromatic ring led to different degrees of potency and selectivity as anti-toxoplasma agents. Among the synthesized analogues, N(6)-(2,4-dimethoxybenzyl)adenosine exhibited the most favorable anti-toxoplasma activity without host toxicity. The binding mode of the synthesized N(6)-benzyladenosine analogues were characterized to illustrate the role of additional hydrophobic effect and van der Waals interaction within an active site of T. gondii adenosine kinase by induced fit molecular modeling.
Assuntos
Adenosina/análogos & derivados , Antiprotozoários , Toxoplasma/efeitos dos fármacos , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Adenosina Quinase/metabolismo , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Modelos Moleculares , Conformação Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Toxoplasma/enzimologiaRESUMO
Adenosine kinase (AK) is a key enzyme in purine metabolism in the ubiquitous intracellular parasite Toxoplasma gondii and is a potential chemotherapeutic target for the treatment of T. gondii infections. To better understand the structure-activity relationship of 6-substituted purine ribosides, the structures of the T. gondii AK-N6,N6-dimethyladenosine (DMA) complex, the AK-DMA-AMP-PCP complex, the AK-6-methyl mercaptopurine riboside (MMPR) complex and the AK-MMPR-AMP-PCP complex were determined to 1.35, 1.35, 1.75 and 1.75 A resolution, respectively. These structures reveal a conformation intermediate between open and closed, with a small lid-domain rotation of 12 degrees . Residues Gly143-X-X-Gly146 undergo torsional changes upon substrate binding, which together with a Gly68-Gly69 switch induces a hinge bending of the lid domain. The intermediate conformation suggests that ATP binding is independent of adenosine binding. Orienting the gamma-phosphate group of ATP into the optimal catalytic position may be the last step before the onset of chemical catalysis and may require the translocation of Arg136 following the complete closure of the lid domain. 6-Substituted purine-nucleoside analogs are accommodated in a hydrophobic cavity. Modification at the N6 or C6 position of the nucleoside would affect the interactions with the surrounding residues and the binding affinity.
Assuntos
Adenosina Quinase/química , Toxoplasma/enzimologia , Adenosina Quinase/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Nucleotídeos de Purina/química , Nucleotídeos de Purina/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS+IFN-gamma-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.